PROJECT SUMMARY In this request for grant transfer, we aim to continue our work defining the signals and mechanisms that mediate connection of newly formed nephrons to the kidney collecting system. Defining mechanisms of tubule interconnection remains incompletely understood and will be essential for engrafting iPScell derived kidney organoids or other stem cell derived kidney replacement tissue. This grant period will be devoted to completing our study of receptors, ligands, and signal transduction pathways that mediate invasive cell behavior and tubule interconnection. Focusing on the regenerating kidney in adult zebrafish as a model of nephron addition, we will use a combination of loss of function mutants, fluorescent transgene reporters, and in situ markers of tissue invasion to define essential signals and effectors of tubule interconnection. The PI, Dr. Iain Drummond, has moved his lab to the Mount Desert Island Biological Laboratory to take on the position of Professor and Director of the Davis Center for Aging and Regeneration. The reasons for the transfer of institution are: 1) The MDI Biological Laboratory is a historical seat of fundamental kidney research, notably the site of Homer Smith's and Frank Epstein's insightful and seminal studies on kidney evolution and epithelial transport. 2) MDI Bio Lab has evolved to be an NIH Center of Research Excellence in Aging and Regeneration, focusing on fundemental studies using genetic model organisms which aligns with the Drummond lab's focus on zebrafish regeneration. Dr. Drummond assumed the position of PI on the MDIBL COBRE grant allowing him to continue and expand his role mentoring young investigators. 3) Under his directorship MDIBL has obtained new, cutting-edge microscopy equipment (Zeiss 980 two photon confocal microscope) that will enable new live imaging approaches to tubule interconnection studies and further developed single cell RNA seq capabilies that will allow completion of kidney regeneration studies. 4) MDIBL has committed support for the PI, students, and a Postdoc that will increase lab productivity and provide more long term stability. 5) Ongoing collaborations with the Jackson Lab on MDI will enable translation of fish studies to mouse models of kidney engraftment. The career plans of the PI are to continue to lead the field in zebrafish kidney development and regeneration and translate fundamental studies of developmental mechanisms and repair pathways to address human AKI and CKD.